RBM7 Protein Purification and Structure Determination to Examine RBM7 Interaction With 7SK snRNA Upon Genotoxic Stress
This is a recording of UCARE 2020/2021 project. The video is submitted to be presented at Student Research Days 2021. The project examines RBM7 protein purification optimization , structural determination using NMR and RNA-Protein interaction using iCLIP.
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[00:00:01.090]Hi, my name is Amr.
[00:00:03.040]Today. I'll be presenting my project
[00:00:04.568]about RBM7 protein purification
[00:00:07.930]and structure determination to examine
[00:00:09.920]RBM7 interaction with 7SK RNA upon genotoxic stress.
[00:00:15.420]First I'll give an introduction about 7SK RNP
[00:00:19.656]and RNA-binding motif 7 protein.
[00:00:22.450]Second, I will discuss my approach
[00:00:24.270]to the questions of the project and the methods I used.
[00:00:27.480]Finally, I'll give the conclusion
[00:00:29.370]and the next steps in the project.
[00:00:32.560]Central dogma, the main theme of molecular biology
[00:00:35.992]explains gene expression as shown in the figure.
[00:00:39.620]DNA, the molecule of life, gets transcribed into mRNA.
[00:00:44.710]The transcription step composed of three steps,
[00:00:47.270]initiation, elongation, and termination.
[00:00:50.590]After transcription, the mRNA gets translated into protein,
[00:00:54.530]which is the molecule needed by cells
[00:00:56.900]for functional and structural roles.
[00:01:00.350]7SK small nuclear ribonucleoproteins
[00:01:04.296]composed of 7SK noncoding RNA
[00:01:08.330]and other proteins attached to that RNA
[00:01:11.180]is found to regulate transcription step in the cell.
[00:01:14.890]As shown in the figure, upon a signal,
[00:01:17.520]the complex gets disintegrated
[00:01:19.780]and the positive transcription elongation factor P-TEFB
[00:01:23.840]gets released from the complex.
[00:01:25.830]The released P-TEFB then goes through the transcription site
[00:01:29.700]and add a phosphate group to the C-terminal domain
[00:01:33.250]of the paused RNA polymerase.
[00:01:35.730]The first relation leads to the activation
[00:01:37.990]of RNA polymerase and the transition
[00:01:40.910]from a paused state to a processive elongation state.
[00:01:45.050]The release of P-TEFB was found to be caused
[00:01:47.810]by various stimuli with different regulatory components.
[00:01:53.147]RNA-binding motif 7 is a sub unit
[00:01:56.210]of the trimeric nuclear exosome targeting complex NEXT,
[00:02:00.660]which was known to be involved
[00:02:03.670]with in the surveillance of non-coding RNA.
[00:02:06.930]RBM7 was recently found to play a regulatory role
[00:02:11.130]in the release of P-TEFB from 7SK RNP.
[00:02:14.400]The tertiary and the secondary structure of RRM
[00:02:17.170]are found in these two figures.
[00:02:20.600]As mentioned before RBM7 was studied
[00:02:23.010]by Barboric and co-workers and found to have regulatory role
[00:02:26.620]in the release of P-TEFB from 7SK RNP.
[00:02:30.476]As shown in the figure, RBM7 was found to be activated
[00:02:33.900]upon geneotoxic signal by a mutagen or ultraviolet light,
[00:02:38.730]which leads to its interaction with other protein
[00:02:41.789]of 7SK RNP leading to the release of P-TEFB.
[00:02:47.005]P-TEFB can then activate our RNA-enabled mRNAs
[00:02:49.950]to undergo processive elongation
[00:02:52.100]for essential genes to counteract
[00:02:55.270]the DNA damage resulted from the mutagen,
[00:02:58.940]leading to the survival of the cell.
[00:03:01.750]The questions to be answered in this project are
[00:03:04.630]what are the optimized buffering conditions
[00:03:06.500]and purification method for RBM7?
[00:03:08.890]How does RBM7 interact with 7SK upon genotoxic stress?
[00:03:13.250]To answer these questions I designed two constructs of RBM7,
[00:03:19.281]RBM7-91 and RBM7-86 that include the RRM of RBM7
[00:03:24.500]and optimize their purification.
[00:03:26.870]The two constructs were used to determine
[00:03:28.940]the optimized structure using NMR.
[00:03:31.800]Also, I constructed a protocol for iCLIP data analysis
[00:03:34.930]to determine the cross-linking sites
[00:03:37.070]of RBM7 with 7SK RNA.
[00:03:40.530]The first step needed to study RBM7
[00:03:43.360]is to construct the plasmid
[00:03:44.827]and to optimize the purification methods
[00:03:46.730]suitable for its stability and better yield.
[00:03:50.040]The purification steps are shown in this figure.
[00:03:53.210]First, transform the plasmid with RBM7
[00:03:56.280]and histidine tag into E.coli.
[00:03:59.713]Grow bacteria and induce expression with IPTG,
[00:04:04.020]Lyse the bacteria and run
[00:04:05.560]the supernatant over affinity column,
[00:04:08.460]run the protein of size exclusion column,
[00:04:10.550]concentrate the protein,
[00:04:12.739]and the last step is to use NMR
[00:04:15.810]for structural identification of RBM7.
[00:04:19.520]One of the optimization step is protein expression
[00:04:22.240]using IPTG inducer.
[00:04:24.330]In this step I used four different conditions
[00:04:26.670]to assess the yield of the purified protein.
[00:04:29.890]These conditions have different IPTG concentration,
[00:04:33.720]expression time and induction temperature.
[00:04:38.010]In condition one and two no protein at the expected size
[00:04:41.380]of RBM7 construct was observed.
[00:04:43.600]On the other hand, condition three and four
[00:04:45.820]showed protein at the expected size.
[00:04:48.990]Condition three was 0.5 millimolar IPTG
[00:04:52.050]and three hours was found to have the best yield
[00:04:55.130]of 460 micromolar and 850 microliter.
[00:05:00.110]Another optimization step is the protein buffer conditions.
[00:05:04.720]Two buffer conditions were used as follows.
[00:05:07.660]The conditions were assessed for their suitability
[00:05:10.380]for RBM7 stability in these buffer condition
[00:05:13.460]using nuclear magnetic resonance NMR.
[00:05:17.790]HSQC NMR for the two RBM7 construct were performed
[00:05:22.610]in the two different buffer conditions.
[00:05:25.300]As shown in the two spectrums,
[00:05:26.540]the left for RBM7-91 in condition one,
[00:05:29.910]the right is for the same construct in condition two.
[00:05:33.200]It's observed that condition one led
[00:05:35.390]to first precipitation of RBM7 protein,
[00:05:38.530]while condition two is found to stabilize
[00:05:40.390]RBM7 for longer time.
[00:05:42.520]This is further confirmed by converting the two spectrum
[00:05:45.330]in which some peaks are not found using condition one,
[00:05:48.770]compared to condition two.
[00:05:51.650]On the other hand, his6-RBM7-86 is found to be
[00:05:55.210]unstable in buffer condition two,
[00:05:58.140]as shown in the NMR spectrum.
[00:05:59.880]Also first disappearance of the peak was observed
[00:06:02.510]using NMR as time goes.
[00:06:04.510]Therefore, RBM7-91 construct in condition two
[00:06:07.840]has better stability and longer shelf life
[00:06:10.110]than RBM7-86 in both conditions.
[00:06:14.420]The final method I used in the project
[00:06:16.300]is iCLIP to determine the cross-linking site
[00:06:19.580]of RBM7 with 7SK RNA upon genotoxic stress
[00:06:23.800]using computational methods.
[00:06:25.720]The raw data was taken from Barboric and co-worker studies
[00:06:28.670]and I developed a protocol to analyze the iCLIP raw data.
[00:06:32.380]The iCLIP experiment has two different treatment,
[00:06:35.030]one with DMSO as a control,
[00:06:36.834]and the other has 4-NQO as a mutagen.
[00:06:39.960]The first graph shows the control without a mutagen
[00:06:43.500]and as shown RBM7 was found to cross link
[00:06:46.410]with 7SK mainly at position 251,
[00:06:50.080]with frequency of about 0.23.
[00:06:52.920]However, the treated experiment shows an increase
[00:06:55.820]in the frequency of the linking sites at the same position
[00:07:00.410]with frequency of about 0.3.
[00:07:02.930]This data is also presented in the heat map here
[00:07:05.480]for the two experiments.
[00:07:08.500]The conclusions of the study are
[00:07:10.890]his6-RBM7-86 is less stable than his6-RBM7-91
[00:07:16.087]in the buffer condition as identified by NMR.
[00:07:18.660]The more cross links of RBM7 to 7SK
[00:07:21.370]is found upon treating with a mutagen,
[00:07:23.680]especially in residue 250 and 251,
[00:07:28.080]reflecting enhanced protein RNA interactions.
[00:07:30.670]The future directions is to run 3D NMR
[00:07:34.050]for double-labeled sample of the RBM7-91,
[00:07:37.330]precisely define binding sites using fluorescent anisotropy,
[00:07:41.690]do in vivo experiment for further downstream
[00:07:44.459]to determine other interactions upon genotoxic stress.
[00:07:48.130]At the end, I would like to thank my research advisor,
[00:07:51.010]Dr. Catherine Eichhorn for her guidance and support.
[00:07:53.750]And I'd like to thank all these people
[00:07:55.600]for making this project possible.
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